Sortases act as transacylases that accept a wide range of substrates. Staphylococcus aureus sortase A and its derivatives recognize an LPXTG motif in their substrates, cleave it between the Thr and Gly residues with concomitant formation of a covalent thioacylenzyme intermediate, and then resolve the intermediate in a nucleophilic attack by a stretch of exposed NH2-terminal Gly residues as part of a modified short peptide or even a protein. Such modification of proteins, referred to as sortagging and further developed in the PI's laboratory, has enabled the installation of a variety of different chemical modifications, including fluorophores, nucleic acids, lipids, click handles, and radioisotopes. This application exploits the unique properties of sortase to address two major unsolved questions in immunobiology at a molecular and at an organismal level, the solutions to which have wide biological and biomedical implications. The dynamics of the B cell receptor for antigen (BCR) have been difficult to explore for lack of reagents that track the BCR without eliciting activation. Cas9/CRISPR genome editing experiments have yielded mice in which the immunoglobulin C? locus has been modified with an in-frame COOH-terminal LPETG motif, so that its ? light chains can be modified site-specifically in a sortase-catalyzed reaction with fluorophores suitable for Frster resonance energy transfer (FRET) measurements and stochastic optical reconstruction microscopy (STORM). Site-specific modification of the BCR at sites away from its antigen binding sites provides an unprecedented opportunity to analyze the nanoscale organization of the BCR on the surface of living B cells, both at rest and in response to antigen. Our model will enable us to explore the fate of the BCR and B cell responses and reconcile published findings that support seemingly contradictory models. Resolving this issue is of fundamental importance to understand how the BCR works. This will be pursued in Aim 1. Non-invasive imaging of an immune response in an intact mouse or human is a key goal to track sites of incipient and ongoing inflammation, to visualize an anti-viral response, or to follo initiation, execution or suppression of an anti-tumor immune response. Camelid-derived single domain antibodies (VHHs) possess a short circulatory t1/2 and superior tissue penetration. Owing to their small size (~15 kDa), VHHs are ideal vehicles for non-invasive imaging of their targets by positron emission tomography (PET). Synthetic routes to the installation of 18F and 64Cu applied to sortase-tag equipped Class II MHC-specific VHHs have yielded the first PET-imaging-ready labeled VHHs. These clearly reveal sites of inflammation, and based on these encouraging preliminary data, this approach will now be extended in Aim 2 to imaging T cells in the course of an immune response both in influenza and tumor models, using newly developed T-cell specific VHHs, labeled with either 18F or 64Cu.